Methods of treating respiratory disorders

ABSTRACT

A method of treating a hyperoxia induced disease or disorder associated with GSNO deficiency in a subject in need thereof includes administering to the subject a therapeutically effective amount of GSNO or a GSNO promoting agent.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/287,515, filed Jan. 27, 2016, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No.K12HD057581-05 and 1P01HL101871 awarded by The National Institutes ofHealth. The United States government has certain rights in theinvention.

TECHNICAL FIELD

This application relates to compositions and methods of modulatingS-nitrosoglutathione (GSNO) in subjects with respiratory disordersassociated with hyperoxia and particularly relates to the use of GSNOreductase inhibitors to treat hyperoxia-induced air responsiveness,respiratory distress syndrome, and bronchopulmonary dysplasia.

BACKGROUND

Globally, more than 11% of babies are born before 37 weeks of gestation(premature), and the number of premature births is increasing worldwide(Blencowe et al., (2012) Lancet 379:2162-2172). Bronchopulmonarydysplasia (BPD) is the major pulmonary morbidity of extreme prematurity,with an estimated 14,000 new diagnoses made each year in the UnitedStates (Van Marter (2009) Semin Fetal Neonatal Med 14:358-366) andannual care costs upwards of $26 billion (Cole et al., (2011) Pediatrics127:363-369). Equally important is the concept that prematurity and BPDmay be a chronic respiratory condition. After their initial care, halfof premature patients will be rehospitalized for respiratory causes inearly childhood (Furman et al., (1996) The Journal of Pediatrics128:447-452). Follow up studies of children and young adults bornprematurely show evidence of impaired pulmonary function, manifestingsigns of obstructive pulmonary disease with decreased predicted forcedexpiratory volume in 1 second (FEV1) (Fawke et al. (2010) Am J RespirCrit Care Med 182:237-247), decreased predicted forced expiratory flow(FEF25-75%) (Vollsaeter et al. (2013) Thorax 68:767-776), and reducedexercise capacity (Vrijlandt et al., (2006) Am J Respir Crit Care Med173:890-896). Indeed, increased airway reactivity and asthma-likesymptoms are common long-term pulmonary consequences of both prematurebirth and BPD (Baraldi et al., (2009) Early Human Development 85:S1-3).

S-nitrosothiols (SNOs) are ubiquitous protein molecules in which nitricoxide is bound to a cysteine thiol, which regulate the biologic activityof many target proteins (Foster et al., (2009) Trends in MolecularMedicine 15:391-404). One such SNO is S-nitrosoglutathione (GSNO), anendogenous bronchodilator, which exhibits 100-fold more potency than theasthma medication theophylline (Gaston et al., (1994) J Pharmacol ExpTher 268:978-984). GSNO is a critical modulator of airway reactivity inasthmatic animal models (Blonder et al., (2014) BMC Pulm Med 14:3).Airway levels of GSNO are dramatically decreased in pediatric cases ofsevere asthmatic respiratory failure (Gaston et al., (1998) Lancet351:1317-1319) and GSNO reductase (GSNOR, also known as alcoholdehydrogenase 5, adh5), the enzyme responsible for the catabolicbreakdown of GSNO, is elevated in asthma patients that display increasedairway reactivity (Que et al. (2009) American Journal of Respiratory andCritical Care Medicine 180:226-231).

Traditional asthma therapies are not always effective in this patientpopulation. Interestingly, the asthma phenotype in premature infantsdiffers from the allergic asthma seen in their term-born peers(Filippone et al., (2013) Eur Respir J 42:1430-1431). The increased riskfor airway reactivity in surviving premature neonates is stronglyassociated with a history of prolonged supplemental oxygen exposure andbronchopulmonary dysplasia (BPD), compared to the reactivity observed infull term peers, which instead is associated with a history of geneticinheritance, allergy, airway inflammation, and cigarette exposures(Halvorsen et al., (2005) Pediatr Allergy Immunol 16:487-494). Yet,former premature infants are twice as likely to be prescribed asthmamedications compared to their full term school-age classmates (Hack etal., (2005) JAMA 294:318-325) and premature infants, with or withoutBPD, continue to be at very high risk for airway reactivity in infancyand childhood (Fawke et al. (2010) Am J Respir Crit Care Med182:237-247; Been et al., (2014) PLoS Med 11:e1001596; Hennessy et al.,(2013) J Pediatr 163:61-66 e61). Thus, novel therapies are needed inthis growing patient population.

SUMMARY

Embodiments described herein relate to the targeted replacement ofdepleted S-nitrosoglutathione (GSNO) stores in the developing lungs ofinfant and child subjects born prematurely and potentially treated withprolonged supplemental oxygen by administration of GSNO directly to thesubject and/or inhibition of S-nitrosoglutathione reductase (GSNOR) inthe subject, and particularly relates to methods of treating and/orpreventing hyperoxia induced respiratory disorders, such as respiratorydistress syndrome or bronchopulmonary dysplasia (BPD), in prematuresubjects in need thereof, including those premature subjects receivinglife saving newborn interventions, such as supplemental oxygen andmechanical ventilation, by administering to the subjects GSNO and/or aGSNO promoting agent.

In some embodiments GSNO or a GSNO promoting agent can be administeredto a subject, such as a premature subject, to raise the subject's GSNOlevels and treat disorders associated with GSNO deficiency, such ashyperoxia induced respiratory disorders associated with prolongedsupplemental oxygen treatment. The GSNO and/or GSNO promoting agent canbe administered to the subject at a therapeutically effective amount(s)in a pharmaceutical composition comprising GSNO and/or a GSNO promotingagent and at least one pharmaceutically acceptable carrier.

Other embodiments described herein relate to methods of treating BPD ina subject in need thereof. Such a method comprises administering atherapeutically effective amount of a pharmaceutical compositionincluding GSNO and/or a GSNO promoting agent and at least onepharmaceutically acceptable carrier.

Still other embodiments described herein relate to methods of treating aBPD, such as hyperoxia induce BPD, in a subject in need thereof byadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a pyrrole inhibitor of GSNOR and at least onepharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-D) illustrate graphs, plots, and western blots showing anincreased GSNOR activity and expression in 3-week-old mice afterneonatal hyperoxia exposure. (A) GSNOR activity was assessed by timedGSNO catabolism in lung homogenates, normalized to protein. GSNORactivity was increased in hyperoxia. Representative nitric oxideanalyzer tracings in triplicate are shown. Data were normallydistributed with equal variance, so a two-tailed Student t test wasused. n=5. *P, 0.05. (B) GSNOR kinetics were estimated by generating aLineweaver-Burke plot at differing GSNO substrate loads. Maximumvelocity/Michaelis-Menton constant did not differ between groups. Datawere normally distributed with equal variance, so a two-tailed Student ttest was used. n=5. (C) Representative Western blot bands from the samegel are shown. Relative expression of GSNOR:b-actin ratio was increasedin hyperoxia. Data were normally distributed with unequal variance, so atwo-tailed Welch's t test was used. n=12. *P, 0.05. (D) RepresentativeWestern blot bands from the same gel are shown. Relative expression ofeNOS:b-actin was increased in hyperoxia. Data were normally distributedwith equal variance, so a two-tailed Student t test was used. n=4. *P,0.05.

FIGS. 2(A-F) illustrate GSNOR immunostaining following hyperoxia inlungs of 3-week old mice. Representative immunohistochemical probe forGSNOR (brown) of inflation-fixed lung sections showed prominent stainingof airway epithelium (arrows) and smooth muscle (*) in the bronchus (A,D) and bronchioles (B, E) of both groups. Sections were counterstainedwith methylene blue. Primary antibody was omitted as a negative control(C, F). Scale bar=50 mm.

FIG. 3 illustrates a graph showing microRNA-342-3p expression in lunghomogenates from 3-week-old mouse pups. Fold decreases in miR-342-3pexpression were observed in hyperoxia compared with room air controls.Data were normally distributed with equal variance, so a two-tailedStudent t test was used. n=6. ***P, 0.001.

FIG. 4 illustrates a western blot and graph showing the results of miRsilencing of GSNOR protein expression in mouse macrophage RAW264.7 cellswere transiently transfected with a miR-342-3p mimic or a miR mimiccontrol (cel-miR-67). Western blot analysis for GSNOR was performed onlysed cells 48 hours after transfection. Representative Western blotbands from the same gel are shown. Relative expression of GSNOR:b-actinratio was decreased in cells overexpressing miR-342-3p. Data werenormally distributed with equal variance, so a two-tailed Student t testwas used. n=8. *P, 0.05.

FIGS. 5(A-B) illustrate plots showing GSNO aerosol or GSNOR inhibitionattenuates hyperoxia-induced airway hyperresponsiveness to methacholinechallenge. Aerosolized methacholine dose responses were compared in (A)3-week-old mouse pups raised from birth in room air (21%) or hyperoxia(60%) and in (B) adult 6-week-old mice raised in room air or recoveredin room air after the initial 3-week hyperoxia exposure. Mice werepretreated with saline vehicle aerosol, 10 mM GSNO aerosol, or 1 mg/kgN6022 GSNOR inhibitor injection. Rrs was significantly increased inhyperoxia at 3 weeks and after room air recovery at 6 weeks of age;pretreatment with GSNO or N6022 attenuated these changes. Comparisonswere made to 21%+saline control. Two-way analysis of variance with fixedsequence Tukey-Kramer post hoc analysis from highest to lowestmethacholine dose was used. *P, 0.05, **P, 0.01, ***P, 0.001.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

It will be noted that the structure of some of the compounds of theapplication include asymmetric (chiral) carbon or sulfur atoms. It is tobe understood accordingly that the isomers arising from such asymmetryare included herein, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. The compounds of thisapplication may exist in stereoisomeric form, therefore can be producedas individual stereoisomers or as mixtures.

The term “isomerism” means compounds that have identical molecularformulae but that differ in the nature or the sequence of bonding oftheir atoms or in the arrangement of their atoms in space. Isomers thatdiffer in the arrangement of their atoms in space are termed“stereoisomers”. Stereoisomers that are not mirror images of one anotherare termed “diastereoisomers”, and stereoisomers that arenon-superimposable mirror images are termed “enantiomers”, or sometimesoptical isomers. A carbon atom bonded to four nonidentical substituentsis termed a “chiral center” whereas a sulfur bound to three or fourdifferent substitutents, e.g. sulfoxides or sulfinimides, is likewisetermed a “chiral center”.

The term “chiral isomer” means a compound with at least one chiralcenter. It has two enantiomeric forms of opposite chirality and mayexist either as an individual enantiomer or as a mixture of enantiomers.A mixture containing equal amounts of individual enantiomeric forms ofopposite chirality is termed a “racemic mixture”. A compound that hasmore than one chiral center has 2n−1 enantiomeric pairs, where n is thenumber of chiral centers. Compounds with more than one chiral center mayexist as either an individual diastereomer or as a mixture ofdiastereomers, termed a “diastereomeric mixture”. When one chiral centeris present, a stereoisomer may be characterized by the absoluteconfiguration (R or S) of that chiral center. Alternatively, when one ormore chiral centers are present, a stereoisomer may be characterized as(+) or (−). Absolute configuration refers to the arrangement in space ofthe substituents attached to the chiral center. The substituentsattached to the chiral center under consideration are ranked inaccordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn etal, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al.,Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London),612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964,41, 116).

The term “geometric Isomers” means the diastereomers that owe theirexistence to hindered rotation about double bonds. These configurationsare differentiated in their names by the prefixes cis and trans, or Zand E, which indicate that the groups are on the same or opposite sideof the double bond in the molecule according to the Cahn-Ingold-Prelogrules. Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof.

The term “atropic isomers” are a type of stereoisomer in which the atomsof two isomers are arranged differently in space. Atropic isomers owetheir existence to a restricted rotation caused by hindrance of rotationof large groups about a central bond. Such atropic isomers typicallyexist as a mixture, however as a result of recent advances inchromatography techniques, it has been possible to separate mixtures oftwo atropic isomers in select cases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The term “derivative” refers to compounds that have a common corestructure, and are substituted with various groups as described herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

“Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a patient at risk for or afflicted with adisease, including improvement in the condition through lessening orsuppression of at least one symptom, delay in progression of thedisease, prevention or delay in the onset of the disease, etc.

The terms “prevent,” “preventing,” or “prevention” are art-recognizedand include precluding, delaying, averting, obviating, forestalling;stopping, or hindering the onset, incidence, severity, or recurrence ofa disease, disorder or condition from occurring in a subject, which maybe predisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it. Preventing a condition related to a diseaseincludes stopping the condition from occurring after the disease hasbeen diagnosed but before the condition has been diagnosed.

The term “pharmaceutical composition” refers to a formulation containingthe disclosed compounds in a form suitable for administration to asubject. In a preferred embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salts thereof) in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, inhalational, and the like. Dosage forms for the topical ortransdermal administration of a compound described herein includespowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, nebulized compounds, and inhalants. In a preferred embodiment,the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The compounds of the application are capable of further forming salts.All of these forms are also contemplated herein.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. For example, the saltcan be an acid addition salt. One embodiment of an acid addition salt isa hydrochloride salt. The pharmaceutically acceptable salts can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrilebeing preferred. Lists of salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990).

The compounds described herein can also be prepared as esters, forexample pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group ina compound can be converted to its corresponding ester, e.g., anacetate, propionate, or other ester.

The compounds described herein can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound,which releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds can bedelivered in prodrug form. Thus, the compounds described herein areintended to cover prodrugs of the presently claimed compounds, methodsof delivering the same and compositions containing the same. “Prodrugs”are intended to include any covalently bonded carriers that release anactive parent drug in vivo when such prodrug is administered to asubject. Prodrugs are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxy, amino, sulfhydryl, carboxy, orcarbonyl group is bonded to any group that may be cleaved in vivo toform a free hydroxyl, free amino, free sulfhydryl, free carboxy or freecarbonyl group, respectively. Prodrugs can also include a precursor(forerunner) of a compound described herein that undergoes chemicalconversion by metabolic processes before becoming an active or moreactive pharmacological agent or active compound described herein.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, ester groups (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds, and thelike, as well as sulfides that are oxidized to form sulfoxides orsulfones.

The term “protecting group” refers to a grouping of atoms that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Examples of protecting groups can be found in Green andWuts, Protective Groups in Organic Chemistry, (Wiley, 2.sup.nd ed.1991); Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski,Protecting Groups, (Verlag, 3^(rd) ed. 2003).

The term “amine protecting group” is intended to mean a functional groupthat converts an amine, amide, or other nitrogen-containing moiety intoa different chemical group that is substantially inert to the conditionsof a particular chemical reaction. Amine protecting groups arepreferably removed easily and selectively in good yield under conditionsthat do not affect other functional groups of the molecule. Examples ofamine protecting groups include, but are not limited to, formyl, acetyl,benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,2-trimethylsilyl-ethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl(NVOC), and the like. Those of skill in the art can identify othersuitable amine protecting groups.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl, and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers.

Additionally, the salts of the compounds described herein, can exist ineither hydrated or unhydrated (the anhydrous) form or as solvates withother solvent molecules. Nonlimiting examples of hydrates includemonohydrates, dihydrates, etc. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The term “solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The compounds, salts and prodrugs described herein can exist in severaltautomeric forms, including the enol and imine form, and the keto andenamine form and geometric isomers and mixtures thereof. Tautomers existas mixtures of a tautomeric set in solution. In solid form, usually onetautomer predominates. Even though one tautomer may be described, thepresent application includes all tautomers of the present compounds. Atautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertible by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs.

Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2.formation of a delocalized anion (e.g., an enolate); 3. protonation at adifferent position of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

The term “analogue” refers to a chemical compound that is structurallysimilar to another but differs slightly in composition (as in thereplacement of one atom by an atom of a different element or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analogue is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

A “patient,” “subject,” or “host” to be treated by methods describedherein may mean either a human or non-human animal, such as a mammal, afish, a bird, a reptile, or an amphibian. Thus, the subject of theherein disclosed methods can be a human, non-human primate, horse, pig,rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term doesnot denote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. In another aspect, the subject is aprematurely born mammal treated with prolonged supplemental oxygen. Apatient refers to a subject afflicted with a disease or disorder.

The terms “prophylactic” or “therapeutic” treatment is art-recognizedand includes administration to the host of one or more of thetherapeutic compositions described herein. If it is administered priorto clinical manifestation of the unwanted condition (e.g., disease orother unwanted state of the host animal) then the treatment isprophylactic, i.e., it protects the host against developing the unwantedcondition, whereas if it is administered after manifestation of theunwanted condition, the treatment is therapeutic (i.e., it is intendedto diminish, ameliorate, or stabilize the existing unwanted condition orside effects thereof).

The terms “therapeutic agent”, “drug”, “medicament” and “bioactivesubstance” are art-recognized and include molecules and other agentsthat are biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in a patient or subject totreat a disease or condition. The terms include without limitationpharmaceutically acceptable salts thereof and prodrugs. Such agents maybe acidic, basic, or salts; they may be neutral molecules, polarmolecules, or molecular complexes capable of hydrogen bonding; they maybe prodrugs in the form of ethers, esters, amides and the like that arebiologically activated when administered into a patient or subject.

The phrase “therapeutically effective amount” or “pharmaceuticallyeffective amount” is an art-recognized term. In certain embodiments, theterm refers to an amount of a therapeutic agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, the term refers to thatamount necessary or sufficient to eliminate, reduce or maintain a targetof a particular therapeutic regimen. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation. Incertain embodiments, a therapeutically effective amount of a therapeuticagent for in vivo use will likely depend on a number of factors,including: the rate of release of an agent from a polymer matrix, whichwill depend in part on the chemical and physical characteristics of thepolymer; the identity of the agent; the mode and method ofadministration; and any other materials incorporated in the polymermatrix in addition to the agent.

The term “ED50” is art-recognized. In certain embodiments, ED50 meansthe dose of a drug, which produces 50% of its maximum response oreffect, or alternatively, the dose, which produces a pre-determinedresponse in 50% of test subjects or preparations. The term “LD50” isart-recognized. In certain embodiments, LD50 means the dose of a drug,which is lethal in 50% of test subjects. The term “therapeutic index” isan art-recognized term, which refers to the therapeutic index of a drug,defined as LD50/ED50.

The terms “IC₅₀,” or “half maximal inhibitory concentration” is intendedto refer to the concentration of a substance (e.g., a compound or adrug) that is required for 50% inhibition of a biological process, orcomponent of a process, including a protein, subunit, organelle,ribonucleoprotein, etc.

With respect to any chemical compounds, the present application isintended to include all isotopes of atoms occurring in the presentcompounds. Isotopes include those atoms having the same atomic numberbut different mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include C-13 and C-14.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent can be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent can be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

When an atom or a chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it is meant to encompass each number within therange as well as all intermediate ranges. For example, “C₁₋₆ alkyl” ismeant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3,1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” is intended to include both branched (e.g., isopropyl,tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl(e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groupshave a specified number of carbon atoms. For example, C₁₋₆ alkyl isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. As usedherein, “lower alkyl” refers to alkyl groups having from 1 to 6 carbonatoms in the backbone of the carbon chain. “Alkyl” further includesalkyl groups that have oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more hydrocarbon backbone carbon atoms. In certainembodiments, a straight chain or branched chain alkyl has six or fewercarbon atoms in its backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ forbranched chain), for example four or fewer. Likewise, certaincycloalkyls have from three to eight carbon atoms in their ringstructure, such as five or six carbons in the ring structure.

The term “substituted alkyls” refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). If not otherwise indicated, the terms “alkyl” and “loweralkyl” include linear, branched, cyclic, unsubstituted, substituted,and/or heteroatom-containing alkyl or lower alkyl, respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally,although again not necessarily, alkenyl groups can contain 2 to about 18carbon atoms, and more particularly 2 to 12 carbon atoms. The term“lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, andthe specific term “cycloalkenyl” intends a cyclic alkenyl group,preferably having 5 to 8 carbon atoms. The term “substituted alkenyl”refers to alkenyl substituted with one or more substituent groups, andthe terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which atleast one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkenyl” and “lower alkenyl” include linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl and lower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The terms “alkyl”, “alkenyl”, and “alkynyl” are intended to includemoieties which are diradicals, i.e., having two points of attachment. Anonlimiting example of such an alkyl moiety that is a diradical is—CH₂CH₂—, i.e., a C₂ alkyl group that is covalently bonded via eachterminal carbon atom to the remainder of the molecule.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “aryl” refers to an aromatic substituent containing a singlearomatic ring or multiple aromatic rings that are fused together,directly linked, or indirectly linked (such that the different aromaticrings are bound to a common group such as a methylene or ethylenemoiety). Aryl groups can contain 5 to 20 carbon atoms, and particularlypreferred aryl groups can contain 5 to 14 carbon atoms. Examples of arylgroups include benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diaryl amino, and alkylaryl amino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl). If nototherwise indicated, the term “aryl” includes unsubstituted,substituted, and/or heteroatom-containing aromatic substituents.

The term “alkaryl” refers to an aryl group with an alkyl substituent,and the term “aralkyl” refers to an alkyl group with an arylsubstituent, wherein “aryl” and “alkyl” are as defined above. Exemplaryaralkyl groups contain 6 to 24 carbon atoms, and particularly preferredaralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groupsinclude, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl,4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl,4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like.Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl,p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,3-ethyl-cyclopenta-1,4-diene, and the like.

The terms “heterocyclyl” or “heterocyclic group” include closed ringstructures, e.g., 3- to 10-, or 4- to 7-membered rings, which includeone or more heteroatoms. “Heteroatom” includes atoms of any elementother than carbon or hydrogen. Examples of heteroatoms include nitrogen,oxygen, sulfur and phosphorus.

Heterocyclyl groups can be saturated or unsaturated and includepyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine,lactones, lactams, such as azetidinones and pyrrolidinones, sultams, andsultones. Heterocyclic groups such as pyrrole and furan can havearomatic character. They include fused ring structures, such asquinoline and isoquinoline. Other examples of heterocyclic groupsinclude pyridine and purine. The heterocyclic ring can be substituted atone or more positions with such substituents as described above, as forexample, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety.Heterocyclic groups can also be substituted at one or more constituentatoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy,a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, or —CN, or the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.“Counterion” is used to represent a small, negatively charged speciessuch as fluoride, chloride, bromide, iodide, hydroxide, acetate, andsulfate. The term sulfoxide refers to a sulfur attached to 2 differentcarbon atoms and one oxygen and the S—O bond can be graphicallyrepresented with a double bond (S═O), a single bond without charges(S—O) or a single bond with charges [S(+)-O(−)].

The terms “substituted” as in “substituted alkyl,” “substituted aryl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: functional groups such as halo, hydroxyl, silyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₄ alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl(—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano (—CN), isocyano (—N⁺C⁻),cyanato (—O—CN), isocyanato (—ON⁺C⁻), isothiocyanato (—S—CN), azido(—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-and di-(C₁-C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino(—PH₂); and the hydrocarbyl moieties C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation, and asappropriate, purification from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The terms “free compound” is used herein to describe a compound in theunbound state.

Throughout the description, where compositions are described as having,including, or comprising, specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the compositionsand methods described herein remains operable. Moreover, two or moresteps or actions can be conducted simultaneously.

The term “small molecule” is an art-recognized term. In certainembodiments, this term refers to a molecule, which has a molecularweight of less than about 2000 amu, or less than about 1000 amu, andeven less than about 500 amu.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

The terms “healthy” and “normal” are used interchangeably herein torefer to a subject or particular cell or tissue that is devoid (at leastto the limit of detection) of a disease condition.

Embodiments described herein relate to the targeted replacement ofdepleted S-nitrosoglutathione (GSNO) stores in the developing lungs ofinfant and child subjects born prematurely, and potentially treated withprolonged supplemental oxygen, by administration of GSNO directly to thesubject and/or inhibition of S-nitrosoglutathione reductase (GSNOR) inthe subject. The lungs of premature infant subjects are underdeveloped,and these infant subjects often need assistance with their breathing andoxygen supplementation. Yet, these life-saving interventions, such assupplemental oxygen and mechanical ventilation, may increase theseinfant's risk for future breathing problems, the most severe of which isbronchopulmonary dysplasia (BDP).

Utilizing a hyperoxic neonatal mouse model of BPD, it was found thatsubjects raised in hyperoxia had increased activity and proteinexpression of the primary catabolic enzyme of S-nitrosoglutathionereductase (GSNOR). It was also found that GSNO supplementation and/orinhibition of GSNOR has protective effects on airway hyperreactivityfound after neonatal oxygen exposure in a mouse model of BPD.

Without being bound by theory, it is believed that an increase in theexpression and catabolic activity of GSNOR, the enzyme responsible forreducing and inactivating GSNO, through altered post-transcriptionalregulation by microRNA-342-3p results in neonatal subjects developingpulmonary toxicities and/or respiratory disorders when treated withprolonged supplemental oxygen. Therefore, it is contemplated thattargeted direct replacement of depleted GSNO stores in the developinglung of subjects exposed to hyperoxia or inhibition of the catabolicactivity of GSNOR can be therapeutically effective in the treatment ofinfant and child subjects born prematurely and that have been treatedwith prolonged supplemental oxygen.

In some embodiments, a method of treating and/or preventing hyperoxiainduced respiratory disorders, such as respiratory distress syndrome orbronchopulmonary dysplasia (BPD), in premature subjects in need thereof,such as a premature or prenatal subject exposed to supplemental oxygentreatment, can include administering to the subject a therapeuticallyeffective amount of GSNO or a GSNO promoting agent.

In some embodiments, GSNO used in the methods described herein caninclude aerolized GSNO this is provided in a pharmaceutical composition,which is administered to the subject via inhalation. For example, GSNOcan be provided in an aerolized pharmaceutical composition at aconcentration of about 1 μM to about 100 mM GSNO (e.g., about 10 mMGSNO) with a pharmaceutically acceptable carrier that is administered tothe subject via inhalation.

The GSNO promoting agent for use in the methods described herein caninclude any agent capable of increasing the expression of GSNO and/orinhibiting the catabolic activity of GSNOR in a subject. Examples ofGSNO promoting agents for use in the methods described herein includeADH inhibitors, such as GSNOR inhibitors, AKR inhibitors, and SNO-CoARinhibitors. Administration of ADH inhibitors, AKR inhibitors, and/orSNO-CoAR inhibitors as well as SNO-CoA (or derivatives thereof e.g.,SNO-cysteamine) to a subject in need thereof can raise GSNO levels inthe subject and treat diseases or disorders associated with GSNOdeficiency, such as BPD, and hyperoxia induced airwayhyperresponsiveness.

In some embodiments, the GSNO promoting agent can be a GSNOR inhibitor(also known as an ADH5 inhibitor). In one example, the GSNOR inhibitorcan include a pyrrole inhibitor of GSNOR. In some embodiments, thepyrrole inhibitor of GSNOR can be a compound having the followingformula:

-   -   wherein Ar is an aryl, such as a phenyl and thiophenyl;    -   R₁ is selected from the group consisting of unsubstituted or        substituted imidazolyl, chloro, bromo, fluoro, hydroxy, and        methoxy;    -   R₂ is selected from the group consisting of hydrogen, methyl,        chloro, fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl,        dimethylamino, amino, formamido, and trifluoromethyl; and    -   X is selected from the group consisting of CO and SO₂; and        pharmaceutically acceptable salts, stereoisomers, prodrug, or        metabolites thereof.

One example of an ADH5 inhibitor/GSNOR pyrrole inhibitor is GSNORpyrrole inhibitor N6022, which is commercially available from NivalisTherapeutics, Boulder, Colo. N6022 has the following formula:

Additional inhibitors of GSNOR for use in methods recited herein aredescribed in U.S. Patent Application Publication Nos: 2011/0136875,2011/0136881, 2011/0144110, 2011/0144180, 2012/0245210, 2013/0253024,2014/0057957, 2014/0113938, 2014/0113945, 2014/0155447, 2014/0194425,2014/0194481 and U.S. Pat. Nos. 8,470,857, 8,642,628, 8,673,961,8,686,015, 8,691,816, 8,759,548, 8,846,736, 8,957,105, 9,029,402,9,138,427, and 9,180,119 all of which are incorporated herein byreference in their entirety.

Other ADH inhibitors that can be used as a GSNO promoting agent caninclude auramine O, allicin, 1,5-anilinonaphthalenesulfonic acid,1,7-anilinonaphthalenesulfonic acid, 1,8-anilinonaphthalenesulfonicacid, berberine, canavanine, 2,2′-diprypyl, imidazole,m-methylbenzamide, 4-methylpyrazole, pyrazole, 4-pentylpyrazole,O-phenanthroline, alrestatin, anthranic acid, O-carboxybenzaldehyde,2,3-dimethylsuccinic acid, ethacrynic acid, isonicotinic acid,phenacemide, quercetin, quercitrin, sorbinil, tetramethyleneglutaricacid, valproic acid, propranolol, 2,2,2-trichloroethanol,4,5-diaminopyrazole and its derivatives and2-ethyl-5-methyl-2H-3,4-diaminopyrazole. See U.S. Patent ApplicationPublication US 2003/0138390, which is incorporated herein by referencein its entirety.

Fomepizole (4-methylpyrazole) is also a competitive inhibitor of ADH.Pyrazole and its 4-substituted derivatives competitively inhibit thebinding of alcohol substrates through the formation of a tightenzyme.NAD⁺.inhibitor complex, in which pyrazole nitrogens interact withboth zinc and NAD⁺. Xie et al., J. Biol. Chem., 272:18558-18563 (1997),herein incorporated by reference.

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is anisomeric and isomeric analogue of NAD, in which the nicotinamide ring islinked to the sugar via a C-glycosyl (C5-C1′) bond. CNAD acts as ageneral dehydrogenase inhibitor but shows unusual specificity andaffinity for liver alcohol dehydrogenase. Goldstein et al., J. Med.Chem., 37:392-9 (1994), herein incorporated by reference.

Still other ADH inhibitors include dimethyl sulfoxide, Perlman andWolff, Science, 160:317-9 (1968); and p-methylbenzyl hydroperoxide,Skursky et al., Biochem Int., 26:899-904 (1992), herein incorporated byreference.

In some embodiments, the ADH inhibitor can be a selective ADH6 inhibitoror partially selective ADH6 inhibitor that does not inhibit ADH3. Inother embodiments, the ADH inhibitor does not inhibit ADH3 but inhibitsother ADHs, such as ADH6.

In some embodiments, the AKR inhibitor can be a selective AKR1A1inhibitor or a partially selective AKR1A1 that can inhibit otheraldo-keto reductase family members, such as AKR1B1. In some embodiments,the AKR1A1 inhibitor can have an IC₅₀≤100 nM. In other embodiments, theAKR1A1 inhibitor can have a selectivity for AKR1A1 versus AKR1B1≥10times. In other embodiments, the AKR1A1 inhibitor can have a selectivityfor AKR1A1 versus other AKRs≥50 times. In still other embodiments, theAKR1A1 inhibitor can have an AKR1A1 IC₅₀≤25 nM and an AKR1B1/AKR1A1IC₅₀≤300 nM (e.g., less than 100 nM).

Examples of selective and partially selective AKR1A1 inhibitors caninclude Imirestat(2,7-Difluoro-2′H,5′H-spiro[fluorene-9,4′-imidazolidine]-2′,5′-dione)and analogues thereof.

In some embodiments, the imirestat analogues can include compoundsselected from the group consisting of:

-   -   each R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are the same or different        and are one or more substituent selected from the group        consisting of hydrogen, halogen, substituted or unsubstituted        C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₃-C₂₀ aryl,        heterocycloalkenyl containing from 5-6 ring atoms, (wherein from        1-3 of the ring atoms is independently selected from N, NH,        N(C₁-C₆ alkyl), NC(O)(C₁-C₆ alkyl), O, and S), heteroaryl or        heterocyclyl containing from 5-14 ring atoms, (wherein from 1-6        of the ring atoms is independently selected from N, NH, N(C₁-C₃        alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl,        hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄        alkynyloxy, C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl        (—CO-alkyl) and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy        (—O-acyl), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀        aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato        (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),        carboxy (—COOH), carboxylato (—COO⁻), carbamoyl (—(CO)—NH₂),        C₁-C₂₄ alkyl-carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl        (—(CO)—NH-aryl), thiocarbamoyl (—(CS)—NH₂), carbamido        (—NH—(CO)—NH₂), cyano (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN),        isocyanato (—O—N⁺═C⁻), isothiocyanato (—S—CN), azido (—N═N⁺═N⁻),        formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄        alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido        (—NH—(CO)-alkyl), C₆-C₂₀ arylamido (—NH—(CO)-aryl), sulfanamido        (—SO2NR2 where R is independently H, alkyl, aryl or heteroaryl),        imino (—CR═NH where R is hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl,        C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino (—CR═N(alkyl),        where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.),        arylimino (—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl,        etc.), nitro (—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato        (—SO₂—O⁻), C₁-C₂₄ alkylsulfanyl (—S-alkyl; also termed        “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”),        C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl        (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀        arylsulfonyl (—SO₂-aryl), sulfonamide (—SO₂—NH2, —SO₂NY₂        (wherein Y is independently H, arlyl or alkyl), phosphono        (—P(O)(OH)₂), phosphonato (—P(O)(O)₂), phosphinato (—P(O)(O⁻)),        phospho (—PO₂), phosphino (—PH₂), polyalkyl ethers (—[(CH₂)_(n)        O]_(m)), phosphates, phosphate esters [—OP(O)(OR)₂ where R═H,        methyl or other alkyl], groups incorporating amino acids or        other moieties expected to bear positive or negative charge at        physiological pH, and combinations thereof; and pharmaceutically        acceptable salts thereof.

Other examples of selective and partially selective AKR1A1 inhibitorscan include Methyl[4-oxo-2-(substituted benzoylimino)-3-(substitutedphenyl)thiazolidin-5-ylidene]acetate derivatives recited in S. Ali etal., “Design, synthesis and molecular modeling of novelmethyl[4-oxo-2-(aroylimino)-3-(substitutedphenyl)thiazolidin-5-ylidene]acetates as potent and selective aldosereductase inhibitors”, Med. Chem. Commun., 2012, 3, 1428-1434. TheseAKR1A1 inhibitors can have the following formula:

-   -   wherein R⁸ and R⁹ are independently selected from the group        consisting of substituted and unsubstituted aryls.

Other examples of selective and partially selective AKR1A1 inhibitorscan include benzothiazolyl substituted iminothiazolidinones andbenzamido-oxothiazolidines recited in Saeed et al., “Benzothiazolylsubstituted iminothiazolidinones and benzamido-oxothiazolidines aspotent and partly selective aldose reductase inhibitors”, Med. Chem.Commun., 2014, 5, 1371-1380. These AKR1A1 inhibitors can have thefollowing formula:

-   -   wherein R¹⁰, R¹¹, or R¹² include one or more substitutent and        are each independently selected from the group consisting of H,        a halogen (e.g., 6-Br, 3-Cl, 2-F, 2-Br, 5,6-di-C1, 2,4-di-C1),        lower alkyl, and methoxy (e.g., 4-OCH₃, 3,4-OCH₃).

Still other examples of selective and partially selective AKR1A1inhibitor are disclosed in the following publications: Mechanism ofHuman Aldehyde Reductase: Characterization of the Active Site Pocket,Oleg A. Barski et al., Biochemistry 1995, 34, 11264-11275, In vivo roleof aldehyde reductase, M. Takahashi et al., Biochim Biophys Acta. 2012November; 1820(11):1787-96, The Aldo-Keto Reductase Superfamily and itsRole in Drug Metabolism and Detoxification, Oleg A. Barski et al., DrugMetab Rev. 2008; 40(4): 553-624, Asborin Inhibits Aldo/Keto Reductase1A1, Matthias Scholz et al., ChemMedChem, 2011, 6, 89-93, Inhibition ofAldehyde Reductase by Aldose Reductase Inhibitors, Sanai Sato et al.,Biochemical Pharmacology, 1990. 40, 1033-1042, Inhibition of humanaldose and aldehyde reductases by non-steroidal anti-inflammatory drugs,D. Michelle Ratliff et al., Advances in Experimental Medicine andBiology, Volume: 463, Issue: Enzymology and Molecular Biology ofCarbonyl Metabolism 7, Pages: 493-499 (1999.), Inhibition of aldehydereductases, Philip J. Schofield et al., Progress in Clinical andBiological Research, 1987, 232, Issue: Enzymol. Mol. Biol. CarbonylMetab., 287-96, Aldose Reductase Inhibitors as Potential TherapeuticDrugs of Diabetic Complications, By Changjin Zhu, DOI: 10.5772/54642,Aldose Reductase Inhibitors: A Potential New Class of Agents for thePharmacological Control of Certain Diabetic Complications, Peter F.Kador et al., Journal of Medicinal Chemistry, 1985, 28, 841-849, Recentclinical experience with aldose reductase inhibitors, H. M. J. Krans,Journal of Diabetes and its Complications, 1992, 6, 39-44, A NovelSeries of Non-Carboxylic Acid, Non-Hydantoin Inhibitors of AldoseReductase with Potent Oral Activity in Diabetic Rat Models:6-(5-Chloro-3-methylbenzofuran-2-sulfonyl)-2H-pyridazin-3-one andCongeners, Banavara L. Mylari et al., J. Med. Chem. 2005, 48, 6326-6339,A Diverse Series of Substituted Benzenesulfonamides as Aldose ReductaseInhibitors with Antioxidant Activity: Design, Synthesis, and in VitroActivity, Polyxeni Alexiou et al., J. Med. Chem. 2010, 53, 7756-7766,Aldose Reductase Inhibitors as Potential Therapeutic Drugs of DiabeticComplications, By Changjin Zhu, DOI: 10.5772/54642, Aldose ReductaseInhibitors: A Potential New Class of Agents for the PharmacologicalControl of Certain Diabetic Complications, Peter F. Kador et al.,Journal of Medicinal Chemistry, 1985, 28, 841-849, Recent clinicalexperience with aldose reductase inhibitors, H. M. J. Krans, Journal ofDiabetes and its Complications, 1992, 6, 39-44, A Novel Series ofNon-Carboxylic Acid, Non-Hydantoin Inhibitors of Aldose Reductase withPotent Oral Activity in Diabetic Rat Models:6-(5-Chloro-3-methylbenzofuran-2-sulfonyl)-2H-pyridazin-3-one andCongeners, Banavara L. Mylari et al., J. Med. Chem. 2005, 48, 6326-6339,A Diverse Series of Substituted Benzenesulfonamides as Aldose ReductaseInhibitors with Antioxidant Activity: Design, Synthesis, and in VitroActivity, Polyxeni Alexiou et al., J. Med. Chem. 2010, 53, 7756-7766,all of which are incorporated herein by reference in their entirety. Itwill be appreciated that any potential selective or partially selectiveAKR1A1 inhibitors can be used in the compositions and methods recitedherein.

In other embodiments, the GSNO promoting agent can include an agent thatreduces or inhibits ADH and/or AKR expression, such as ADH6 expressionor AKR1A1 expression, in tissue or cells of a subject in need thereof.“Expression”, means the overall flow of information from a gene toproduce a gene product (typically a protein, optionallypost-translationally modified or a functional/structural RNA).

In some embodiments, the agent can include an RNAi construct thatinhibits or reduces expression of the ADH and/or AKR expression in acell. RNAi constructs comprise RNA that can specifically blockexpression of a target gene. “RNA interference” or “RNAi” is a terminitially applied to a phenomenon observed in plants and worms wheredouble-stranded RNA (dsRNA) blocks gene expression in a specific andpost-transcriptional manner.

As used herein, the term “dsRNA” refers to small interfering RNA (siRNA)molecules or other RNA molecules including a double stranded feature andable to be processed to siRNA in cells, such as hairpin RNA moieties.

The term “loss-of-function,” as it refers to genes inhibited by thesubject RNAi method, refers to a diminishment in the level of expressionof a gene when compared to the level in the absence of RNAi constructs.

As used herein, the phrase “mediates RNAi” refers to (indicates) theability to distinguish which RNAs are to be degraded by the RNAiprocess, e.g., degradation occurs in a sequence-specific manner ratherthan by a sequence-independent dsRNA response, e.g., a PKR response.

As used herein, the term “RNAi construct” is a generic term usedthroughout the specification to include small interfering RNAs (siRNAs),hairpin RNAs, and other RNA species, which can be cleaved in vivo toform siRNAs. RNAi constructs herein also include expression vectors(also referred to as RNAi expression vectors) capable of giving rise totranscripts which form dsRNAs or hairpin RNAs in cells, and/ortranscripts which can produce siRNAs in vivo.

“RNAi expression vector” (also referred to herein as a “dsRNA-encodingplasmid”) refers to replicable nucleic acid constructs used to express(transcribe) RNA which produces siRNA moieties in the cell in which theconstruct is expressed. Such vectors include a transcriptional unitcomprising an assembly of (1) genetic element(s) having a regulatoryrole in gene expression, for example, promoters, operators, orenhancers, operatively linked to (2) a “coding” sequence which istranscribed to produce a double-stranded RNA (two RNA moieties thatanneal in the cell to form an siRNA, or a single hairpin RNA which canbe processed to an siRNA), and (3) appropriate transcription initiationand termination sequences.

The choice of promoter and other regulatory elements generally variesaccording to the intended host cell. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer to circular double stranded DNA loops, which, intheir vector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, theapplication describes other forms of expression vectors that serveequivalent functions and which become known in the art subsequentlyhereto.

The RNAi constructs contain a nucleotide sequence that hybridizes underphysiologic conditions of the cell to the nucleotide sequence of atleast a portion of the mRNA transcript for the gene to be inhibited(i.e., the “target” gene). The double-stranded RNA need only besufficiently similar to natural RNA that it has the ability to mediateRNAi. Thus, embodiments tolerate sequence variations that might beexpected due to genetic mutation, strain polymorphism or evolutionarydivergence. The number of tolerated nucleotide mismatches between thetarget sequence and the RNAi construct sequence is no more than 1 in 5basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50basepairs. Mismatches in the center of the siRNA duplex are mostcritical and may essentially abolish cleavage of the target RNA. Incontrast, nucleotides at the 3′ end of the siRNA strand that iscomplementary to the target RNA do not significantly contribute tospecificity of the target recognition.

Sequence identity may be optimized by sequence comparison and alignmentalgorithms known in the art and calculating the percent differencebetween the nucleotide sequences by, for example, the Smith-Watermanalgorithm as implemented in the BESTFIT software program using defaultparameters (e.g., University of Wisconsin Genetic Computing Group).Greater than 90% sequence identity, or even 100% sequence identity,between the inhibitory RNA and the portion of the target gene ispreferred. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the target gene transcript.

Production of RNAi constructs can be carried out by chemical syntheticmethods or by recombinant nucleic acid techniques. Endogenous RNApolymerase of the treated cell may mediate transcription in vivo, orcloned RNA polymerase can be used for transcription in vitro. The RNAiconstructs may include modifications to either the phosphate-sugarbackbone or the nucleoside, e.g., to reduce susceptibility to cellularnucleases, improve bioavailability, improve formulation characteristics,and/or change other pharmacokinetic properties. For example, thephosphodiester linkages of natural RNA may be modified to include atleast one of a nitrogen or sulfur heteroatom. Modifications in RNAstructure may be tailored to allow specific genetic inhibition whileavoiding a general response to dsRNA. Likewise, bases may be modified toblock the activity of adenosine deaminase. The RNAi construct may beproduced enzymatically or by partial/total organic synthesis, a modifiedribonucleotide can be introduced by in vitro enzymatic or organicsynthesis.

Methods of chemically modifying RNA molecules can be adapted formodifying RNAi constructs (see for example, Nucleic Acids Res,25:776-780; J Mol Recog 7:89-98; Nucleic Acids Res 23:2661-2668;Antisense Nucleic Acid Drug Dev 7:55-61). Merely to illustrate, thebackbone of an RNAi construct can be modified with phosphorothioates,phosphoramidate, phosphodithioates, chimericmethylphosphonate-phosphodiesters, peptide nucleic acids,5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g.,2′-substituted ribonucleosides, a-configuration).

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount, which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition, while lower doses may also be useful for specificapplications. Inhibition is sequence-specific in that nucleotidesequences corresponding to the duplex region of the RNA are targeted forgenetic inhibition.

In certain embodiments, the subject RNAi constructs siRNAs. Thesenucleic acids are around 19-30 nucleotides in length, and even morepreferably 21-23 nucleotides in length, e.g., corresponding in length tothe fragments generated by nuclease “dicing” of longer double-strandedRNAs. The siRNAs are understood to recruit nuclease complexes and guidethe complexes to the target mRNA by pairing to the specific sequences.As a result, the target mRNA is degraded by the nucleases in the proteincomplex. In a particular embodiment, the 21-23 nucleotides siRNAmolecules comprise a 3′ hydroxyl group.

The siRNA molecules described herein can be obtained using a number oftechniques known to those of skill in the art. For example, the siRNAcan be chemically synthesized or recombinantly produced using methodsknown in the art. For example, short sense and antisense RNA oligomerscan be synthesized and annealed to form double-stranded RNA structureswith 2-nucleotide overhangs at each end (Proc Natl Acad Sci USA,98:9742-9747; EMBO J, 20:6877-88). These double-stranded siRNAstructures can then be directly introduced to cells, either by passiveuptake or a delivery system of choice, such as described below.

In certain embodiments, the siRNA constructs can be generated byprocessing of longer double-stranded RNAs, for example, in the presenceof the enzyme dicer. In one embodiment, the Drosophila in vitro systemis used. In this embodiment, dsRNA is combined with a soluble extractderived from Drosophila embryo, thereby producing a combination. Thecombination is maintained under conditions in which the dsRNA isprocessed to RNA molecules of about 21 to about 23 nucleotides.

The siRNA molecules can be purified using a number of techniques knownto those of skill in the art. For example, gel electrophoresis can beused to purify siRNAs. Alternatively, non-denaturing methods, such asnon-denaturing column chromatography, can be used to purify the siRNA.In addition, chromatography (e.g., size exclusion chromatography),glycerol gradient centrifugation, affinity purification with antibodycan be used to purify siRNAs.

In certain embodiments, the RNAi construct is in the form of a hairpinstructure (named as hairpin RNA). The hairpin RNAs can be synthesizedexogenously or can be formed by transcribing from RNA polymerase IIIpromoters in vivo. Examples of making and using such hairpin RNAs forgene silencing in mammalian cells are described in, for example, GenesDev, 2002, 16:948-58; Nature, 2002, 418:38-9; RNA, 2002, 8:842-50; andProc Natl Acad Sci, 2002, 99:6047-52. Preferably, such hairpin RNAs areengineered in cells or in an animal to ensure continuous and stablesuppression of a desired gene. It is known in the art that siRNAs can beproduced by processing a hairpin RNA in the cell.

An RNAi construct for use in a method described herein can include asmall non-coding RNA molecule known as microRNA (miRNA) which functionsin RNA silencing and post-transcriptional regulation of gene expression.In an exemplary embodiment, miRNA can include miR-342-3p or a miR-342-3pmimic to silence transcription and protein expression of GSNOR in asubject.

In yet other embodiments, a plasmid can be used to deliver thedouble-stranded RNA, e.g., as a transcriptional product. In suchembodiments, the plasmid is designed to include a “coding sequence” foreach of the sense and antisense strands of the RNAi construct. Thecoding sequences can be the same sequence, e.g., flanked by invertedpromoters, or can be two separate sequences each under transcriptionalcontrol of separate promoters. After the coding sequence is transcribed,the complementary RNA transcripts base-pair to form the double-strandedRNA.

PCT application WO01/77350 describes an example of a vector forbi-directional transcription of a transgene to yield both sense andantisense RNA transcripts of the same transgene in a eukaryotic cell.Accordingly, certain embodiments provide a recombinant vector having thefollowing unique characteristics: it comprises a viral replicon havingtwo overlapping transcription units arranged in an opposing orientationand flanking a transgene for an RNAi construct of interest, wherein thetwo overlapping transcription units yield both sense and antisense RNAtranscripts from the same transgene fragment in a host cell.

In some embodiments, a lentiviral vector can be used for the long-termexpression of a siRNA, such as a short-hairpin RNA (shRNA), to knockdownexpression of the GSNOR in a lung tissue cells of a subject in needthereof. Although there have been some safety concerns about the use oflentiviral vectors for gene therapy, self-inactivating lentiviralvectors are considered good candidates for gene therapy as they readilytransfect mammalian cells.

By way of example, short-hairpin RNA (shRNA) down regulation of theAKR1A1 expression can be created using OligoEngene software(OligoEngine, Seattle, Wash.) to identify sequences as targets of siRNA.The oligo sequences can be annealed and ligated into linearized pSUPERRNAi vector (OligoEngine, Seattle, Wash.) and transformed in E colistrain DH5α cells. After positive clones are selected, plasmid can betransfected into 293T cells by calcium precipitation. The viralsupernatant collected containing shRNA can then be used to infectmammalian cells in order to down regulate the AKR1A1.

AKR1A1 siRNA, shRNA plasmids, and shRNA lentiviral particle genesilencers are commercially available from Santa Cruz Biotechnology underthe product names sc-78566, sc-78566-SH, and sc-78566-V.

In another embodiment, the ADH and/or AKR inhibitor can includeantisense oligonucleotides. Antisense oligonucleotides are relativelyshort nucleic acids that are complementary (or antisense) to the codingstrand (sense strand) of the mRNA encoding a particular protein.Although antisense oligonucleotides are typically RNA based, they canalso be DNA based. Additionally, antisense oligonucleotides are oftenmodified to increase their stability.

The binding of these relatively short oligonucleotides to the mRNA isbelieved to induce stretches of double stranded RNA that triggerdegradation of the messages by endogenous RNAses. Additionally,sometimes the oligonucleotides are specifically designed to bind nearthe promoter of the message, and under these circumstances, theantisense oligonucleotides may additionally interfere with translationof the message. Regardless of the specific mechanism by which antisenseoligonucleotides function, their administration to a cell or tissueallows the degradation of the mRNA encoding a specific protein.Accordingly, antisense oligonucleotides decrease the expression and/oractivity of a particular protein (e.g., AKR1A1 or GSNOR/ADH5).

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups, such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Proc Natl Acad Sci 86:6553-6556; Proc Natl Acad Sci84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents (See,e.g., BioTechniques 6:958-976) or intercalating agents. (See, e.g.,Pharm Res 5:539-549). To this end, the oligonucleotide may be conjugatedor coupled to another molecule.

Oligonucleotides described herein may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Bio search, Applied Biosystems, etc.).As examples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209), methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Proc Natl Acad Sci 85:7448-7451).

The selection of an appropriate oligonucleotide can be performed by oneof skill in the art. Given the nucleic acid sequence encoding aparticular protein, one of skill in the art can design antisenseoligonucleotides that bind to that protein, and test theseoligonucleotides in an in vitro or in vivo system to confirm that theybind to and mediate the degradation of the mRNA encoding the particularprotein. To design an antisense oligonucleotide that specifically bindsto and mediates the degradation of a particular protein, it is importantthat the sequence recognized by the oligonucleotide is unique orsubstantially unique to that particular protein. For example, sequencesthat are frequently repeated across protein may not be an ideal choicefor the design of an oligonucleotide that specifically recognizes anddegrades a particular message. One of skill in the art can design anoligonucleotide, and compare the sequence of that oligonucleotide tonucleic acid sequences that are deposited in publicly availabledatabases to confirm that the sequence is specific or substantiallyspecific for a particular protein.

A number of methods have been developed for delivering antisense DNA orRNA to cells; e.g., antisense molecules can be injected directly intothe tissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antisense oligonucleotide sufficient to suppress translation onendogenous mRNAs in certain instances. Therefore, another approachutilizes a recombinant DNA construct in which the antisenseoligonucleotide is placed under the control of a strong pol III or polII promoter. For example, a vector can be introduced in vivo such thatit is taken up by a cell and directs the transcription of an antisenseRNA. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells.

Expression of the sequence encoding the antisense RNA can be by apromoter known in the art to act in mammalian, preferably human cells.Such promoters can be inducible or constitutive. Such promoters includebut are not limited to: the SV40 early promoter region (Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Cell 22:787-797), the herpes thymidine kinasepromoter (Proc Natl Acad Sci 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Nature 296:39-42), etc. A type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct that can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systematically).

In some embodiments, GSNO and/or the GSNO promoting agent can beprovided in pharmaceutical compositions with at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-inventive compound active agents.

Pharmaceutical compositions comprising GSNO and/or the GSNO promotingagent can be utilized in any pharmaceutically acceptable dosage form,including, but not limited to injectable dosage forms, liquiddispersions, gels, aerosols, ointments, creams, lyophilizedformulations, dry powders, tablets, capsules, controlled releaseformulations, fast melt formulations, delayed release formulations,extended release formulations, pulsatile release formulations, mixedimmediate release and controlled release formulations, etc.

Specifically, GSNO and/or the GSNO promoting agent can be formulated:(a) for administration selected from the group consisting of oral,pulmonary, intravenous, intra-arterial, intrathecal, intra-articular,rectal, ophthalmic, colonic, parenteral, intracisternal, intravaginal,intraperitoneal, local, buccal, nasal, and topical administration; (b)into a dosage form selected from the group consisting of liquiddispersions, gels, aerosols, ointments, creams, tablets, sachets, andcapsules; (c) into a dosage form selected from the group consisting oflyophilized formulations, dry powders, fast melt formulations,controlled release formulations, delayed release formulations, extendedrelease formulations, pulsatile release formulations, and mixedimmediate release and controlled release formulations; or (d) anycombination thereof.

For targeted delivery to a subject's airway or lung tissue in accordancewith a method described above, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry powder or aerosolized or vaporized solutions, dispersions,or suspensions capable of being dispensed by an inhaler or nebulizerinto the endobronchial or nasal cavity of a subject in need thereof. Insome embodiments, GSNO and/or the GSNO promoting agent can be deliveredyea aerosol spray from pressured container or dispenser that contains asuitable propellant, e.g., a gas such as carbon dioxide, a nebulizedliquid, or a dry powder from a suitable device.

By way of example, an aerosol of 10 mM GSNO, at a dose of 0.05 ml/kg,can be delivered to the subject's airway on a daily basis. Additionalembodiments include 1) the use of a similar concentration by drypowdered inhaler, and 2) delivery of greater concentrations to the lowerrespiratory tract by an aerosol bronchoscopy, 3) by atomizer to thenasal mucosa and osteomeatal complex, 4) to the eustachian tube. Thisdosing has as its objective restoring or increasing normal levels ofGSNO to the airway of a subject in need thereof.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,or other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates,or phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes, or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions for injectable use may comprise sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating at least one compound of the invention into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of acompound of the invention plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the compound of the invention can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In some embodiments, GSNO and/or a GSNO promoting agent can be preparedwith carriers that will protect against rapid elimination from the body.For example, a controlled release formulation can be used, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the compounds of the invention may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also include suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of thecompound of the invention calculated to produce the desired therapeuticeffect in association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on the unique characteristics of the compound ofthe invention and the particular therapeutic effect to be achieved, andthe limitations inherent in the art of compounding such an active agentfor the treatment of individuals.

Pharmaceutical compositions that include GSNO and/or a GSNO promotingagent can comprise one or more pharmaceutical excipients. Examples ofsuch excipients include, but are not limited to binding agents, fillingagents, lubricating agents, suspending agents, sweeteners, flavoringagents, preservatives, buffers, wetting agents, disintegrants,effervescent agents, and other excipients. Such excipients are known inthe art. Exemplary excipients include: (1) binding agents which includevarious celluloses and cross-linked polyvinylpyrrolidone,microcrystalline cellulose, silicified microcrystalline cellulose, gumtragacanth and gelatin; (2) filling agents such as various starches,lactose, lactose monohydrate, and lactose anhydrous; (3) disintegratingagents such as alginic acid, Primogel, corn starch, lightly crosslinkedpolyvinyl pyrrolidone, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starchglycolate, and mixtures thereof; (4) lubricants, including agents thatact on the flowability of a powder to be compressed, include magnesiumstearate, colloidal silicon dioxide, talc, stearic acid, calciumstearate, and silica gel; (5) glidants such as colloidal silicondioxide; (6) preservatives, such as potassium sorbate, methylparaben,propylparaben, benzoic acid and its salts, other esters ofparahydroxybenzoic acid such as butylparaben, alcohols such as ethyl orbenzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose; lactose such as lactose monohydrate, andlactose anhydrous; dibasic calcium phosphate, mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, bubble gum flavor, fruit flavors, and the like; and (10)effervescent agents, including effervescent couples such as an organicacid and a carbonate or bicarbonate. Suitable organic acids include, forexample, citric, tartaric, malic, fumaric, adipic, succinic, and alginicacids and anhydrides and acid salts. Suitable carbonates andbicarbonates include, for example, sodium carbonate, sodium bicarbonate,potassium carbonate, potassium bicarbonate, magnesium carbonate, sodiumglycine carbonate, L-lysine carbonate, and arginine carbonate.Alternatively, only the sodium bicarbonate component of the effervescentcouple may be present.

In some embodiments, pharmaceutical compositions comprising GSNO and/ora GSNO promoting agent can be used for prophylactic therapy. Forexample, a therapeutically effective amount of GSNO and/or a GSNOpromoting agent can be administered to a premature subject in needthereof prior to oxygen exposure or during daily oxygen exposure toprevent the subject from developing BPD.

In certain embodiments, subjects can include neonatal human patientsexperiencing treatment with prolonged supplemental oxygen. Neonatalpatients commonly develop wheezing disorders and bronchopulmonarydysplasia BPD following such prolonged targeted oxygen supplementationtreatment necessary for neonatal survival.

While premature infants with BPD have the most severe lung disease, itis further contemplated that all premature infants, even those thoughtto be late-preterm or near term, are at significantly increased risk forwheezing and asthma than their full-term peers. Therefore, in certainembodiments, subjects administered therapeutic GSNO repletingcompositions in accordance with a method described herein can furtherinclude premature infants without BPD who can benefit from GSNORinhibition.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg/kg to 10 g/kg body weight and often ranges from 10μg/kg to 1 g/kg or 10 μg/kg to 100 mg/kg body weight of the subjectbeing treated, per day. In a particular embodiment, the therapeuticallyeffective amount of the GSNOR inhibitor, N6022, ranges from 1 mg/kg to100 mg/kg body weight daily.

In some embodiments, the therapeutically effective amount is the amountrequired to decrease by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or even completely reverse airway hyperreactivityobserved in a subject with BPD. In some embodiments, the therapeuticallyeffective amount can be the amount required to increase lung compliance(Crs) at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, ormore and/or decrease lung respiratory resistance (Rrs) associated withhyperoxia in a premature human subject administered supplemental oxygenby at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, ormore. In some embodiments, the therapeutically effective amount can bethe amount required to produce a measurable increase (e.g., at least 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) in forcedexpiratory volume in 1 second (FEV1), forced expiratory flow (FEF2)and/or increased exercise capacity in the subject.

In some embodiments, GSNO and/or a GSNO promoting agent can beadministered in combination with other NO donors, including SNO-CoA,which is shown to have novel activity in regulating sterol biosynthesisand CoA metabolism. An NO donor donates nitric oxide or a related redoxspecies and more generally provides nitric oxide bioactivity, that isactivity which is identified with nitric oxide, e.g., vasorelaxation orstimulation or inhibition of a receptor protein, e.g., ras protein,adrenergic receptor, NFκB. NO donors including S-nitroso, O-nitroso,C-nitroso, and N-nitroso compounds and nitro derivatives thereof andmetal NO complexes, but not excluding other NO bioactivity generatingcompounds, useful herein are described in “Methods in Nitric OxideResearch,” Feelisch et al. eds., pages 71-115 (J. S., John Wiley & Sons,New York, 1996), which is incorporated herein by reference. NO donorswhich are C-nitroso compounds where nitroso is attached to a tertiarycarbon which are useful herein include those described in U.S. Pat. No.6,359,182 and in WO 02/34705. Examples of S-nitroso compounds, includingS-nitrosothiols useful herein other than GSNO, include, for example,S-nitroso-N-acetylpenicillamine, S-nitroso-cysteine and ethyl esterthereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy(N-nitrosamine), andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor. GSNO and/or GSNO promoting agents can also be combined with R(+)enantiomer of amlodipine, a known NO releaser (Zhang at al., J.Cardiovasc. Pharm. 39: 208-214 (2002)).

In some embodiments, GSNO and/or a GSNO promoting agent can beadministered in a combinatorial therapy or combination therapy thatincludes administration of the GSNO and/or a GSNO promoting agent withone or more additional active agents. The phrase “combinatorial therapy”or “combination therapy” embraces the administration of GSNO and/or aGSNO promoting, and one or more therapeutic agents as part of a specifictreatment regimen intended to provide beneficial effect from theco-action of these therapeutic agents. Administration of thesetherapeutic agents in combination typically is carried out over adefined period (usually minutes, hours, days or weeks depending upon thecombination selected). “Combinatorial therapy” or “combination therapy”is intended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example by administering to thesubject an individual dose having a fixed ratio of each therapeuticagent or in multiple, individual doses for each of the therapeuticagents. Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissue. Thetherapeutic agents can be administered by the same route or by differentroutes. The sequence in which the therapeutic agents are administered isnot narrowly critical.

In some embodiments, GSNO and/or GSNO promoting agents can beadministered in combination with active agents, such as vasodilators,prostanoid agonists, antiandrogens, cyclosporins and their analogues,antimicrobials, triterpenes, alone or as a mixture. The vasodilators caninclude potassium channel agonists including minoxidil and itsderivatives, aminexil and the compounds described in U.S. Pat. Nos.3,382,247, 5,756,092, 5,772,990, 5,760,043, 5,466,694, 5,438,058,4,973,474, chromakalin and diazoxide. The antiandrogens can include5α-reductase inhibitors such as finasteride and the compounds describedin U.S. Pat. No. 5,516,779, cyprosterone acetate, azelaic acid, itssalts and its derivatives, and the compounds described in U.S. Pat. No.5,480,913, flutamide and the compounds described in U.S. Pat. Nos.5,411,981, 5,565,467 and 4,910,226. The antimicrobial compounds caninclude selenium derivatives, ketoconazole, triclocarban, triclosan,zinc pyrithione, itraconazole, pyridine acid, hinokitiol, mipirocine,and the compounds described in EP 680745, clinycine hydrochloride,benzoyl or benzyl peroxide and minocycline. The anti-inflammatory agentscan include inhibitors specific for Cox-2 such as for example NS-398 andDuP-697 (B. Batistini et al., DN&P 1994; 7(8):501-511) and/or inhibitorsof lipoxygenases, in particular 5-lipoxygenase, such as for examplezileuton (F. J. Alvarez & R. T. Slade, Pharmaceutical Res. 1992;9(11):1465-1473).

Other active compounds, which can be present in pharmaceuticalcompositions include aminexil and its derivatives,60-[(9Z,12Z)octadec-9,12-dienoyl]hexapyranose, benzalkonium chloride,benzethonium chloride, phenol, oestradiol, chlorpheniramine maleate,chlorophyllin derivatives, cholesterol, cysteine, methionine, benzylnicotinate, menthol, peppermint oil, calcium panthotenate, panthenol,resorcinol, protein kinase C inhibitors, prostaglandin H synthase 1 orCOX-1 activators, or COX-2 activators, glycosidase inhibitors,glycosaminoglycanase inhibitors, pyroglutamic acid esters,hexosaccharidic or acylhexosaccharidic acids, substituted ethylenearyls,N-acylated amino acids, flavonoids, derivatives and analogues ofascomycin, histamine antagonists, triterpenes, such as ursolic acid andthe compounds described in U.S. Pat. Nos. 5,529,769, 5,468,888,5,631,282, saponins, proteoglycanase inhibitors, agonists andantagonists of oestrogens, pseudopterins, cytokines and growth factorpromoters, IL-1 or IL-6 inhibitors, IL-10 promoters, TNF inhibitors,vitamins, such as vitamin D, analogues of vitamin B12 and panthotenol,hydroxy acids, benzophenones, esterified fatty acids, and hydantoin.

It will also be appreciated that certain selective GSNO promoting agentsthat inhibit some ADHs, AKRs, and/or SNO-CoARs can be administered incombination with other selective ADH inhibitors, AKR inhibitors, and/orSNO-CoAR inhibitors that inhibit other ADHs, AKRs, and/or SNO-CoARs. Forexample, a selective GSNOR/ADH5 inhibitor can be administered incombination with an ADH3 inhibitor.

The invention is further illustrated by the following examples, which isnot intended to limit the scope of the claims.

EXAMPLE

In this Example, we show that increased GSNOR activity underlies theperinatal airway hyperreactivity observed in BPD and thus GSNO repletioncan treat perinodal airway hyperreactivity and BPD. We show theseresults in a hyperoxic murine model of BPD and airway hyperreactivity.Murine lung development continues postnatally and is similar to thepremature human lung. Hyperoxia exposure in neonatal mice creates alesion very similar to human BPD with characteristic long-term alveolarand parenchymal remodeling, manifesting increased airway reactivity. Weused this model to investigate the role of GSNOR in BPD airwayhyperreactivity. We demonstrate that neonatal hyperoxia increases GSNORexpression and activity, in part through a microRNA (miR), andGSNO-based treatments can abolish BPD airway hyperreactivity.

Materials and Methods

Animal Hyperoxic Exposure

Animal protocols were approved by the Institutional Animal Care and UseCommittee at Case Western Reserve University (Cleveland, Ohio). Timedpregnant C57BL/6 mice (Charles River Laboratories, Wilmington, Mass.)were maintained on 12-hour light-dark cycles with ad libitum standardfood and water. Within 24 hours of birth, litters were pooled andrandomized into exposure groups. Paired with a nursing dam, pups wereraised in 60% oxygen or room air (21%) for 21 days. Hyperoxia-exposedanimals were housed in standard cages placed in a 38-L Plexiglas chamberwith a continuous flow of blended oxygen (2 L/min). Oxygenconcentrations were monitored twice daily via an oxygen analyzer (miniOXI; MSA Medical, Gurnee, Ill.). To control for oxygen exposures, nursingdams were rotated between paired litters during weekly cage changes.Ventilator studies were conducted and/or tissue harvested within 24hours of removal from hyperoxia at 3 weeks. A subgroup of animals wasreturned to room air following 3 weeks of initial hyperoxia exposure andsubsequently recovered to 6 weeks of age for adult lung mechanic andGSNOR activity studies.

GSNOR Activity by Copper Cysteine Reagent and Nitric Oxide Analysis

Enzyme activity in lung homogenates from 3-week old and 6-week-old micewas assessed by timed GSNO catabolism and quantification bycopper-cysteine reagent and nitric oxide analysis (2C/NOA). Afterterminal anesthesia with i.p. ketamine/xylazine (Pfizer, St. Joseph,Mo.; Lloyd Laboratories, Shenandoah, Iowa), lungs from mice wereharvested and rinsed in ice-cold phosphate-buffered saline (PBS, pH7.4), placed in centrifuge tubes, snap-frozen in liquid nitrogen, andstored at 280° C. Tissue in ice-cold radioimmunoprecipitation assaylysis buffer containing protease inhibitors (Santa Cruz Biotechnology,Dallas, Tex.) was homogenized, and protein levels were quantified byPierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific,Waltham, Mass.). A known quantity of GSNO (28 mM) was loaded withcoreagents (300 mM NADH and 2 mM glutathione; Sigma-Aldrich, St. Louis,Mo.) and equivalent protein quantities of frozen lung homogenates inPBS. After incubation for 5 minutes at 37° C., the reaction was quenchedby a 1:10 dilution of ice-cold PBS. Uncatabolized GSNO was then measuredby 2C/NOA, as previously described in detail (Rogers et al., 2013).Briefly, samples were injected into a temperature-controlled reservoircontaining copper cysteine reagent (pH, 6.9) with a continuous flow ofblended helium. Gas-phase nitric oxide was liberated from GSNO containedin the injected samples and detected by ozone-based chemiluminescenceusing an inline nitric oxide analyzer (Seivers 280i; GE Instruments,Boulder, Colo.). GSNO content was determined by fittingchemiluminescence peaks to a GSNO standard curve and normalizing tosample protein levels. Enzyme kinetics were further derived from aLineweaver-Burke double-reciprocal plot utilizing a total of threeloading doses of GSNO (14, 28, and 56 mM).

Western Blot

Harvested snap-frozen lungs from 3-week-old mice were homogenized inice-cold radioimmunoprecipitation assay lysis buffer containing proteaseinhibitors (Santa Cruz Biotechnology), and protein levels weredetermined by BCA assay (Thermo Scientific). Samples of 50 mg proteinwere separated by electrophoresis with 4-15% Mini Protean TGX precastgels (Bio-Rad Laboratories, Hercules, Calif.) and transferred tonitrocellulose membranes (P:0 on iBlot; Invitrogen, Rehovot, Israel).Membranes were blocked with 5% milk or bovine serum albumin (BSA;Sigma-Aldrich) and incubated in GSNOR primary antibody (1:1000 in milk;observed band 40 kDa; Proteintech, Rosemont, Ill.), endothelial NOSprimary antibody (1:1000 in BSA; observed band 140 kDa; BD TransductionLaboratory, San Jose, Calif.), iNOS primary antibody (1:1000 in BSA;observed band 145 kDa, Abcam, Cambridge, Mass.), or neuronalNOS primaryantibody (1:500 in milk; expected band 161 kDa; Abcam) overnight at 4°C. and then horseradish peroxidase-conjugated secondary anti-rabbitantibody (1:3000; Santa Cruz, Dallas, Tex.) or anti-mouse antibody(1:3000; Santa Cruz), as appropriate, for 1 hour at room temperature. Asa loading control, membranes were stripped (Pierce Restore; ThermoScientific) and reprobed with b-actin primary antibody (1:2000 in milk;observed band 42 kDa, Abcam) and anti-mouse horseradishperoxidase-conjugated secondary antibody (1:5000; Abcam). Bandintensities were quantified and normalized to b-actin using Super SignalWest Pico Chemiluminescent Substrate (Thermo Scientific). Relativeintensities were measured using densitometry software (Image J, NIH).

Immunohistochemistry

After terminal anesthesia (ketamine/xylazine), lungs of 3-week-old micewere inflated with intratracheal 10% formalin at 25 cm H₂O; tissue wassaline perfused with PBS (pH 7.4) and then formalin. The right lung waspostfixed in 10% formalin at 4° C. for >24 hours, tissue was paraffinembedded, and 5-mm-cut sections were processed. Tissue sections wereimmunoblotted with GSNOR primary antibody (1:200; Proteintech) at 4° C.overnight and then biotinylated goat anti-rabbit secondary antibody(1:10,000; Vector Laboratories, Burlingame, Calif.) using Vectastain ABCkit, and next counterstained with methylene blue (Sigma-Aldrich), aspreviously described (Marozkina et al., 2012). Primary antibody wasomitted as a negative control. Airways were similarly imaged (Rolera XRCCD camera; Q Imaging, Surrey, Canada).

miR Microarray

RNA was extracted from saline-perfused snapfrozen lungs of 3-week-oldmice preserved in RNAlater-ICE reagent using a miRVana column isolationkit (Life Technologies, Carlsbad, Calif.). RNA was quantified byNanodrop spectroscopy (Thermo Scientific), and microarray analysis ofall mature mouse probes from the miRBase V21 library were comparedbetween groups (LC Sciences, Houston, Tex.). Utilizing a gene-miRinteraction search (Dweep et al., 2011, 2014) for the 39 untranslatedregion binding site of GSNOR mRNA (gene id: adh5, alcohol dehydrogenase5), the most predicted miR candidates were cross-referenced with themicroarray results, and high-probability miRs were selected andconfirmed by quantitative reverse-transcription polymerase chainreaction (qRT-PCR).

qRT-PCR

RNA was extracted from frozen lungs of 3-week-old mice using TRIzolreagent (Life Technologies) and quantified by Nanodrop spectroscopy(Thermo Scientific). cDNA was generated from 1 mg RNA by reversetranscription using qScript cDNA synthesis kit (Quanta Biosciences,Gaithersburg, Md.). Real-time quantitative polymerase chain reaction wasperformed on a StepOne PCR system (Applied Biosystems, Foster City,Calif.) using TaqMan probes (Life Technologies) for GSNOR(Mm00475804_g1) compared with 25% diluted b-actin control (ThermoScientific) with PerfeCTa qPCR FastMix, UNG, ROX (Quanta Biosciences,Gaithersburg, Md.). For microRNA qRT-PCR, RNA was similarly extracted asin the miR microarray studies. cDNA was generated using TaqManprimerspecific assays and MicroRNA Reverse Transcription kit, andrealtime quantitative polymerase chain reaction was performed usingTaqMan MicroRNA assays for microRNA-342-3p (2260, Thermo Scientific)compared with snRNA-U6 control (001973, Thermo Scientific) with TaqManUniversal Master Mix, No AmpErase UNG (Life Technologies). Fold changesare reported utilizing 2{circumflex over ( )}-ddCT method and StepOnesoftware v2.3 (Applied Biosystems).

Transfection with mmu-miR-342-3p and Cytomix Activation of RAW 264.7Cells

RAW 264.7 macrophage cells (American Type Culture Collection, Manassas,Va.) were cultured in Gibco Dulbecco's modified Eagle's medium with 10%fetal bovine serum and 1% penicillin-streptomycin (Life Technologies).Cells were transfected with 20 nM miRIDIAN miR mimic for mmu-miR-342-3por with a miR mimic transfection control, cel-miR-67 (Dharmacon, GELifesciences, Lafayette, Colo.) by AMAXA electroporation utilizingNucleofector Kit V (Lonza Group, Basel, Switzerland), per manufacturer'sinstructions. After 48 hours, cells were harvested for protein or RNAstudies, and pellets were snap frozen. Protein levels were determined byBCA assay, and protein was equivalently loaded for gel electrophoresisand Western blot analysis of GSNOR:b-actin, as described above. RAW264.7 qRT-PCR for miR-342-3p was similarly performed on transfectedcells, as described above, to confirm increased gene expressionresulting from transfection. Additionally, untransfected RAW 264.7 cellsin culture media were incubated with cytomix (10 ng/mL eachinterleukin-1b, tumor necrosis factor-a, interferon-g, andlipopolysaccharide; Sigma-Aldrich) or vehicle for 10 hours to measurechanges in GSNOR expression in the activated macrophage (Tan et al.,2013).

Synthesis of GSNO

GSNO was synthesized in-house. Briefly, using a nitrogen sparge at 4° C.in light-protective conditions, reduced L-glutathione (2 g) in purgedhydrochloric acid (2 N) and purged ultrapure water was S-nitrosylatedwith sodium nitrite (455 mg) over 30-60 minutes (Sigma-Aldrich). Theresulting pink GSNO solution was vacuum filtered, mixed with 10 mLpurged 50% acetone for 10-20 minutes, and filtered again. Samples werelyophilized and stored at 280° C. Concentration was confirmed by Savilleassay.

GSNOR Inhibitor Administration

N6022 is a selective small molecule reversible inhibitor of GSNOR.Powdered N6022 (Nivalis Therapeutics, Boulder, Colo., purchased throughMedChem Express, Monmouth Junction, N.J.) was reconstituted in sterilePBS (pH 7.4) and administered to hyperoxia exposed mice as a single 1mg/kg i.p. injection the day prior to testing lung mechanics.

Lung Mechanics

Under general anesthesia (i.p. ketamine/xylazine), mice were placedsupine on a heated surgical table, tracheostomized, and ventilated via a19-gauge blunt-tip cannula with a commercial rodent ventilator(flexiVent; SCIREQ, Montreal, Canada). Animals were paralyzed (i.p.pancuronium bromide; Sigma-Aldrich) and ventilated at default settings:tidal volume of 10 mL/kg, a rate of 150 breaths/min, a positive endexpiratory pressure of 3 cm H₂O, and a FiO₂ of 50%. Following tworecruitment deep inflations of sustained inspiration up to a pressure of30 cm H₂O for 3 seconds, 10 mM GSNO or saline vehicle was aerosolizedover 10 seconds using an ultrasonic nebulizer (Aeroneb; SCIREQ) divertedinto the ventilator's inspiratory flow. Inhaled GSNO concentration waschosen based upon the published studies in ventilated guinea pigs andhuman trials in cystic fibrosis. After 5 minutes had elapsed, tworecruitment deep inflations were again delivered, and increasingmethacholine doses of 0, 12.5, 25, 50, 100, and 200 mg/mL were similarlyaerosolized over 10 seconds to generate a dose-response curve. Usingcomputer software (flexiWare 5.1, Version 7.2, SCIREQ), fivemeasurements of respiratory system resistance (Rrs) were calculated by a2.5 Hz single-frequency forced oscillation maneuver (Snapshot 150), andan average was reported for each methacholine dose. Respiratorymechanics were measured in both 3-week-old mice immediately followingsustained hyperoxia exposure and separate 6-week-old mice that wererecovered in room air following the initial 3 weeks of hyperoxiaexposure.

Statistics

Data are expressed as means±S.E.M. A minimum of two litters orexperiments was used for each study; n represent individual animals orcell transfections. Data containing two groups were first tested fornormality and variance and then analyzed by two sample student t test,Welch's t test, or Mann-Whitney U test, as appropriate. For multiplecomparisons, analysis of variance with Tukey-Kramer post hoc test wasused. Alterations in airway reactivity with increasing doses ofmethacholine were compared by two-way analysis of variancerepeated-measures analysis with Tukey-Kramer post hoc comparisons usinga fixed-sequence method from highest to lowest methacholine dose. P,0.05 was considered statistically significant.

Materials

If not otherwise stated, all reagents and chemicals were purchased fromSigma-Aldrich and were of an analytical grade.

Results

GSNO Catabolism is Increased after Neonatal Hyperoxia

Increased expression of GSNOR causes loss of the endogenousbronchodilator, GSNO, and increased bronchial hyperreactivity. Using2C/NOA, we have shown that GSNOR activity (NADH-dependent GSNOcatabolism/min/mg protein) in the lungs of 3-week-old mice raised inneonatal hyperoxia was higher than that of room air controls (FIG. 1A).The Lineweaver-Burke plots of estimated maximum velocity andMichaelis-Menton constant tended to be increased among thehyperoxia-exposed group (FIG. 1B), yet the ratio of maximumvelocity/Michaelis-Menton constant was similar between groups. Althoughthese kinetic findings could indicate loss of a noncompetitiveinhibitor, the most likely explanation was increased GSNOR expression inhyperoxia. GSNOR activity was also measured by 2C/NOA in the lunghomogenates from 6-week-old mice who were exposed to 3 weeks ofhyperoxia and then recovered in room air. GSNOR activity remainedsignificantly increased in the hyperoxia-exposed roomair-recovered mice,compared with 6-week-old room air controls (11.84±0.22 versus 11.08±0.17mM/min/mg protein, respectively, P, 0.05), albeit with less catabolicactivity permgprotein than at 3 weeks of age.

GSNOR Expression is Increased after Neonatal Hyperoxia

Consistent with the GSNOR kinetic data in 3-week-old mice, the relativeprotein expression of GSNOR was increased in the lungs of 3-week-oldmice raised in hyperoxia when compared with room air controls, asassessed by Western blot (FIG. 1C).

eNOS Expression is Increased after Neonatal Hyperoxia

The relative protein expression of eNOS was increased in the lungs of3-week-old mice raised in hyperoxia when compared with room aircontrols, as assessed by Western blot (FIG. 1D). iNOS expression was notsignificantly different between groups, and neuronal NOS was notdetected in the lungs of either group by this Western blot preparation(data not shown).

GSNOR Gene Expression is not Increased after Neonatal Hyperoxia

To determine whether differences in GSNOR expression weretranscriptionally mediated, we performed qRT-PCR on lung homogenatesfrom 3-week-old mice raised in hyperoxia or room air. GSNOR mRNAexpression did not differ between groups.

GSNOR Immunohistochemistry

GSNOR immunostaining was prominent in the hyperoxia-exposed 3-week-oldmice and, consistent with previous findings, staining was localized tothe epithelium and smooth muscle of the airways (FIG. 2).

mmu-miR-342-3p Gene Expression is Decreased after Neonatal Hyperoxia

Because GSNOR mRNA expression did not explain differences in GSNORprotein expression, we next investigated whether microRNA gene silencingregulates its expression. Microarray analysis performed on lunghomogenates from individual 3-week-old animals identified miR candidatesfound to have decreased expression in hyperoxia. MicroRNA candidateswere then cross-referenced with the highest predicted gene-miRinteractions to adh5, the GSNOR gene. mmu-miR-342-3p showed trendstoward decreased expression in hyperoxia by microarray and was predictedby six different prediction data sets to interact with the 39untranslated region of GSNOR. mmu-miR-342-3p was confirmed by qRT-PCR tobe significantly underexpressed in the lungs of hyperoxia exposed3-week-old mice when compared with room air controls (FIG. 3).

Transfection with miR-342-3p Decreases GSNOR Expression

We next showed that miR-342-3p decreases protein expression of GSNORusing mouse macrophage RAW 264.7 cells. RAW 264.7 cells endogenouslyexpress GSNOR (confirmed by Western blot in naive cells and thoseactivated with cytomix; no significant difference was observed withcytomix treatment) and were one of the original cell lines used toisolate and describe GSNOR (Liu et al., 2001). RAW 264.7 cellstransiently transfected with a miR-342-3p mimic had decreased GSNORprotein expression compared with cells transfected with a miR mimiccontrol (FIG. 4). We confirmed miR-342-3p overexpression followingtransfection by qRT-PCR.

Hyperoxic Changes in Respiratory Mechanics are Attenuated byPretreatment with a GSNO Aerosol or by GSNOR Inhibition

We have shown that neonatal hyperoxia increased GSNOR activity andexpression. Therefore, we tested whether GSNO repletion or GSNORinhibition could reverse the airway hyperreactivity observed in our BPDmodel. Responses to methacholine-provoked airway hyperresponsivenesswere characterized by measuring Rrs, an indicator of airwayhyperreactivity. Compared with room air controls, 3-week-old mice raisedin hyperoxia displayed elevated Rrs in response to aerosolizedmethacholine challenge (FIG. 5A). Pretreatment of the hyperoxia-exposedmice with a 10-second GSNO aerosol attenuated these changes, such thatthis group was no longer significantly different from room air controls,except at the highest methacholine dose (200 mg/mL). Pretreatment of thehyperoxia-exposed mice with an i.p. injection of a selective inhibitorof GSNOR activity, N6022, attenuated these changes as well, such thatthis group was no longer significantly different from room air controlsat all methacholine doses. Next we show in room air-recoveredsix-week-old animals that neonatal hyperoxia-exposed mice continued tohave elevated Rrs in response to aerosolized methacholine challenge whencompared with room air-raised controls (FIG. 5B). In these six-week-oldmice exposed to neonatal hyperoxia, both pretreatment with GSNOaerosolization and GSNOR inhibition with N6022 remained effective inattenuating the hyperoxia induced airway hyperresponsiveness. At bothages, pretreatment with GSNO in room air-exposed mice did notsignificantly change Rrs when compared with room air saline-treatedcontrols (data not shown), and baseline Rrs prior to aerosolizations wasnot statistically different between groups (data not shown).

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, we claim:
 1. A method of treatingbronchopulmonary dysplasia (BPD) in a subject, the method comprising:administering to the subject a therapeutically effective amount of apyrrole inhibitor of GSNO reductase, wherein the subject is aprematurely born neonatal human subject exposed to supplemental oxygentreatment, and wherein the pyrrole inhibitor of GSNO reductasecomprising a compound having the formula:

and pharmaceutically acceptable salts thereof.
 2. The method of claim 1,wherein the pyrrole inhibitor of GSNO reductase is administered to thesubject via intraperitoneal administration.
 3. The method of claim 1,wherein the therapeutically effective amount of the pyrrole inhibitor ofGSNO reductase is an amount effective to increase the level of GSNO inthe subject's lung tissue.